It is well known that after marrow ablation there is an osteogenic phase where trabeculae of primary bone replace the blood clot and fill the marrow space. The trabeculae are then subjected to osteoclastic resorption that precedes the appearance of regenerated normal marrow. Not only is there osteogenic reaction locally in the marrow cavity, there is stimulation of bone formation in cortical osteons and enhancement of osteo- and chondrogenesis in distant skeletal sites. Observations in mandibular condyles during the osteogenic phase of postablation healing of tibial marrow suggested that the enhanced osteogenesis resulted from an increase in both the number and activity of osteoblasts. It has been proposed that a factor or factors are produced locally by the regenerating marrow that mediate the peripheral osteogenic response after their release into the blood circulation. Bab I. et al., (1988) Endocrinology 123:345; Bab. I. et al., (1985) Calcif Tissue Int 37:551.
The present invention establishes that regenerating bone marrow produces growth factor activity with an effect on osteogenic cells. Additionally, the present invention provides a novel osteogenic growth polypeptide, identified from regenerating bone marrow, which (i) has a stimulatory effect on osteoblastic cells, and (ii) promotes in vivo bone formation.
The novel osteogenic growth polypeptide of the present invention has sequence homology with histone H4, a 102 amino acid protein, and with a fragment of histone H4. Kayne P. S. et al., (1988) Cell 55:27-39; Kharchenho E. P., et al., (1987) Biull. Eksp. Biol. Med. 103(4): 418-420. The references, however, do not disclose polypeptides within the scope of the present invention and do not disclose any of the biological properties of the polypeptides of present invention.
Bone marrow transplantation (BMT) is progressively and rapidly becoming the treatment of choice in instances of hematological malignancies such as lymphomas, Hodgkins's disease and acute leukemia as well as solid cancers, in particular melanoma and breast cancer. Potentially, with improved methods, BMT can also be used for treating other catastrophic diseases--AIDS, aplastic anemia and autoimmune disorders. The aim of all BMT is to replace the host hemopoietic stem cells, totipotent and pluripotent, injured by chemotherapy, radiation or disease. These stem cells can replicate repeatedly and differentiate to give rise to the whole variety of cells present in blood-erythrocytes, platelets and white blood cells which include lymphocytes, monocytes and neutrophils. Resident macrophages and osteoclasts are also derived from hemopoietic totipotent stem cells. As the stem cells differentiate, they commit themselves more and more to a particular lineage until they can form only one kind of the above cells.
The most common way currently available for acquiring enough stem cells for transplantation is to extract one liter or more of marrow tissue from multiple sites in the donor's bones with needle and syringe, an involved process that usually requires general anesthesia. The donors of allogeneic BMT are usually siblings whose tissue types are compatible and sometimes unrelated donors who are matched to the recipient by HLA typing. Autologous transplants, that eliminate the need for HLA matching, may be used in patients undergoing ablative chemoradiotherapy for the eradication of solid tumors. Autologous stem cells may also be obtained from the umbilical cord blood at birth and stored for future administration.
After transplantation and prior to the establishment of a donor-derived functioning marrow the patients hosting BMT present with a transient marked pancytopenia that exposes them to infections. The incidence of bacterial and fungal infection correlates with both the severity and duration of pancytopenia [Slavin S. and Nagler A., (1992) Transplantation]. The recipient must therefore receive a steady supply of fresh red cells, platelets and antibiotics for several weeks until the transplanted stem cells begin producing large quantities of mature blood elements. In instances of allogenic BMT the recipient immune system must be sufficiently suppressed so that it will not reject the transplanted stem cells. At the same time, the transplanted donor's immune system may give rise to graft versus host disease (GVHD) and cause lethal tissue and organ damage. All these considerations dictate prolonged and expensive hospitalization.
BMT would be much more effective if a way could be found to accelerate the process of engraftment, enhance marrow reconstruction, reduce the medical hurdles and shorten the hospitalization period and the incidence of infection, morbidity and mortality [Gabrilove J. L., et al. (1988) N. Engl. J. Med. 318:1414]. The currently available clinical (experimental) treatment for stimulating post BMT marrow reconstruction consists mainly of the administration of recombinant human granulocyte colony stimulating factor (rhG-CSF) and/or recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) [Blazar R. B., et al. (1989) Blood 74:2264]. These cytokines affect directly the proliferation of transplanted pluripotent cells already committed to the white-cell lineages [Vellenga E., et al. (1987) Leukemia 1:584] and consequently decrease the time to leukocyte and neutrophil recovery.
There are, however, some major concerns regarding the therapeutic use of rhG-CSF and rhGM-CSF. Tumors and leukemic cells possess normal receptors for these cytokines [Vellenga E., et al. (1987) Leukemia 1:584] and their administration can increase relapse rates by enhancing the proliferation of residual host tumor cells. Another concern about using CSFs in the setting of BMT is that the CSFs, by stimulating the proliferation of relatively committed cells with no capacity for self renewal, deplete progenitor cell number [Slavin S. and Nagler A., (1992) Transplantation]. For a similar reason, the CSFs fail to support erythropoiesis and platelet formation.
Polypeptides that support hemopoiesis may prove useful in other ways as well. Some investigators have found that adding stem cells from the peripheral blood to those from the bone marrow significantly increases the rate of engraftment. Extracting sufficient numbers of stem cells from peripheral blood is a complicated procedure. Administering such polypeptides to donors to increase the number of stem cells in the blood will improve the feasibility of transplanting stem cells from peripheral blood [Gold D. W., (1991) Sci. Am., December:36].
A prerequisite for hemopoiesis and therefore successful BMT is the presence of functional stromal cells and tissue that comprise the hemopoietic microenvironment, determine the homing of the injected stem cells from the circulation to the bone marrow and support hemopoiesis [Watson J. D. and McKenna J. J., (1992) Int. J. Cell Cloning 10:144]. Marrow derived stromal tissue also provide the conditions to sustain stem cells in in vitro long-term bone marrow cultures. At present this technology suffices to keep stem cells alive. Adding the appropriate hemopoietic polypeptides to these cultures may help expand the stem cell population in vitro, thus providing increased numbers of these cells for transplantation. The combined in vitro-in vivo approach may provide the basis for a forward-looking strategy for (i) obtaining small stem cell preparations from donors' blood or marrow and (ii) healthy individuals to have their stem cells stored for a time when the cells might be needed to treat a serious disease, thus bypassing the complexity associated with the use of allogeneic BMT.
It would therefore be of therapeutic importance to find small peptides that stimulate post BMT hemopoietic reconstruction by enhancing in vivo and/or in vitro the hemopoietic microenvironment of which fibrous tissue, bone and bone cells are important components. Such peptides may also support hemopoiesis in spontaneous occurring or induced myelosuppression conditions that do not necessarily involve BMT.
Preablation therapy using molecules with an OGP-like activity is likely to enhance the hemopoietic microenvironment and consequently stimulate hemopoiesis at the noncommitted stem cell level avoiding the stem cell depiction and white cell discrimination.